EP0041189A1 - Process for the production of Interferon II and stimulated clones for carrying out the process - Google Patents

Process for the production of Interferon II and stimulated clones for carrying out the process Download PDF

Info

Publication number
EP0041189A1
EP0041189A1 EP81103918A EP81103918A EP0041189A1 EP 0041189 A1 EP0041189 A1 EP 0041189A1 EP 81103918 A EP81103918 A EP 81103918A EP 81103918 A EP81103918 A EP 81103918A EP 0041189 A1 EP0041189 A1 EP 0041189A1
Authority
EP
European Patent Office
Prior art keywords
clones
interferon
cell
type
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP81103918A
Other languages
German (de)
French (fr)
Other versions
EP0041189B1 (en
Inventor
Peter Dr. Krammer
Holger Prof.Dr. Kirchner
Fabrizio Dr. Marcucci
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Deutsches Krebsforschungszentrum DKFZ
Original Assignee
Deutsches Krebsforschungszentrum DKFZ
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE3019621A external-priority patent/DE3019621C2/en
Priority claimed from DE19813104900 external-priority patent/DE3104900A1/en
Application filed by Deutsches Krebsforschungszentrum DKFZ filed Critical Deutsches Krebsforschungszentrum DKFZ
Priority to AT81103918T priority Critical patent/ATE3877T1/en
Publication of EP0041189A1 publication Critical patent/EP0041189A1/en
Application granted granted Critical
Publication of EP0041189B1 publication Critical patent/EP0041189B1/en
Expired legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/57—IFN-gamma

Definitions

  • the subject of the invention is a process for the production of type II interferon from T lymphocytes by sensitization and selection of clones from heterogeneous T lymphocyte populations, characterized in that these cell clones are subjected to mitogenic stimulation and after further growth of the stimulated clones type II interferon, in particular is harvested from the cell-free supernatants of the cultures.
  • type II interferon-producing cell clones for performing this method which are obtained from T-lymphocytes by sensitization and selection of clones from heterogeneous T-lymphocyte populations, characterized in that these cell clones are then subjected to mitogenic stimulation.
  • Interferon is produced by the body's own cells and has recently received increased attention as it inhibits the resynthesis of viruses. It will also discusses whether interferon has a therapeutic effect not only in viral but also in tumor diseases, and there are reasons to say so.
  • Type I is produced in cells after virus infections, e.g. from leukocytes and fibroblasts as well as lymphoblasts (Kirchner et al., Pharmacie in our time, 8, 113, 1979).
  • type II interferon is obtainable from immunocytes (Kirchner et al., Immunobiology 156, 65, 1979 and Kirchner et al., Eur. J. Immunol. 10: 224, 1980).
  • type II interferon is significantly more effective against tumors than type I interferon.
  • interferon Although the first work on interferon was published in 1957, manufacturing is still a problem. An essential prerequisite for the experimental and clinical use of interferon, however, is its pure presentation based on the production in sufficient quantity. So far, there has been no satisfactory way of doing this for type II interferon.
  • the subject of the invention now provides a process for the production of type II interferon on a large scale and permits the attainment of increased concentrations of type II interferon in the solutions obtained in the production.
  • the interferon-producing cells are normal cells and not tumor cells, the object being achieved essentially by selecting clones from heterogeneous populations and the selected clones can be stimulated with mitogenic stimulants.
  • homogeneous mass cultures are grown from the individual clones and these T cell clones are subjected to mitogenic stimulation.
  • the mitogenic stimulation of the T cell clones is carried out with different T cell mitogens or different concentrations of the mitogenic stimulating agent are used.
  • the cell clones obtained after the stimulation are cultivated separately.
  • the cell clones are stimulated in serum-free medium.
  • T-lymphocytes thymus-dependent lymphocytes from lymphoid organs (lymph nodes or spleen) or the blood are sensitized in tissue culture. This sensitization can take place, for example, through antigens or T cell mitogens, such as concanavalin A or phytohemagglutinin. Concanavalin A in a concentration of 5 ⁇ g / ml has proven to be particularly favorable when seeding 5 x 10 6 lymph node cells / ml (4 ml cultures).
  • the sensitized T lymphocytes are cleaned after a 2 to 3 day culture and growth factors are added. These growth factors are obtained from the culture supernatant of rat splenic lymphocytes sensitized with 5 ⁇ g Cöncanavalin A / ml (5.x 10 6 cells / ml, 50 ml cultures). This procedure causes some T lymphocytes to start growing continuously as normal cells. The continuous growth of T-lymphocytes depends on the presence and the amount of growth factors in the culture. Such T-lymphocyte cell lines are then cloned in microtiter plates with the addition of irradiated feed cells (eg mouse peritoneal cells). From the individual cloning, homogeneous mass cultures are obtained as descendants of single cells that can be cultivated further in larger bottles and grow continuously. These clones represent a spectrum of functionally heterogeneous normal T lymphocytes in permanent tissue culture.
  • T cell clones have either been found functionally inactive or they have retained the primarily induced specific activity, e.g. had been induced by antigen stimulation. It was not known that the T cell clones could be brought to the expression of functional activities by further mitogenic stimulation. Experiments in particular are negative. that had the goal of generating increased proliferative growth in these T cell clones by incubation with mitogens. Therefore, the idea of producing interferon type II by mitogen stimulation of the T cell clones is not an obvious one, but a major innovation.
  • This process allows large amounts of type II interferon to be produced; in addition, the concentration of type II interferon in the solutions obtained can be increased more than 10 times compared to mass cultures of mixed cells, which is important not only for the absolute amount obtained, but also for the pure presentation or extraction of concentrated preparations.
  • AKR / J mice were, as described, Krammer et al., J. Exp. Med. 151: 1166, 1980, sensitized to the abdominal skin with 2 ⁇ 10 ⁇ l of 30% trinitrochlorobenzene in acetone. 5 days after sensitization, the lymphocytes of the draining axillary and inguinal lymph nodes were brought into culture (5 ⁇ 10 6 cells / ml; 4 ml cultures; Falcon bottles No. 3013, upright) and for 4 to 5 days in a 95% air 5% C0 2 mixture incubated. This leads to a T cell proliferation without antigen restimulation feration, with maximum activity on day 4 to 5.
  • the surviving cells were then purified and cloned as described (PH Krammer, J. Exp. Med. 147: 25, 1978) by Ficoll density centrifugation (Ficoll-Hypaque 1,077 g / cm 3 ).
  • the static mean as described (Eichmann et al., J. Exp. Med. 152: 477, 1980) was a cell / hole of 96-well plates (Falcon No. 3040) on a 5 ⁇ 10 lawn 4 feed cells (peritoneal extrudate cells from the AKR / J mouse) sown.
  • the cloning was carried out in the presence of full medium containing 10% T cell growth factor.
  • the growth factor-containing supernatant was obtained from rat spleen lymphocytes, stimulated in tissue culture for 24 hours with concanavalin A (5 ⁇ 10 6 / ml; 50 ml; 5 ⁇ g Con A / ml; Falcon bottles No. 3024 F, lying) (Eichmann et al., J Exp. Med. 152: 477, 1980).
  • the cells in the 96-well plates were fed twice a week with fresh medium. After about 3 weeks, macroscopically visible T cell clones were found, which were further grown in large bottles (Falcon No. 3024 F) in the same medium.
  • This example is an application example in humans.
  • Lymphocytes were separated from peripheral blood by Ficoll density centrifugation.
  • the cells thus obtained were activated with phythaemagglutinin (PHA, 1%), purified after 2 days of culture via Ficoll and then cloned by means of a statistical average of 1 to 50 cells / hole from 96-hole plates (Falcon No. 3040) on a Lawn from 2 x 10 4 irradiated feed cells (mouse peritoneal cells) and human peripheral blood cells (3 x 10 4 ) was sown. The cloning was carried out in the presence of 20% T cell growth factor and 1% full medium containing PHA.
  • PHA phythaemagglutinin
  • the growth factor-containing supernatant was obtained from human lymphocytes stimulated in tissue culture for 24 hours with phytohemagglutinin (5 x 10 6 / ml; 50 ml; 1% PHA / ml; Falcon bottles No. 3024F, lying).
  • the cells in the 96-well plates were washed once a week with fresh medium and fed feeding cells. After about 3 weeks, macroscopically visible T cell clones were shown.
  • Cells of the individual clones in 0.2 ml growth factor-free medium were then stimulated with 20 ⁇ g / ml PHA, e.g. according to example 1.
  • This step can, even when applied to human cells, also in serum-free medium, e.g. RPMI 1640 can be performed. 24 hours later, the cell-free supernatants from these cultures were harvested and tested for interferon activity in the usual way.
  • the cultures with high interferon titer were then grown to macro cultures in growth medium and PHA-containing complete medium in the presence of irradiated human feed cells.
  • type II interferon-producing cells also makes it possible to use the interferon-producing genes or the genetic material of the selected clones used for interferon production in a manner known per se in bacteria or tumor cells (in tissue culture) for obtaining interferon, which enables an additional process for the large-scale production of type II interferon.
  • PHA phytohemagglutinin
  • an amount of 5 to 30 ⁇ g / ml, in particular 15 to 25, preferably 10 to 20 ⁇ g / ml, has been found to be expedient as concentrations in the stimulation of the clones proven.
  • clones is, of course, to be understood in a broad sense and also includes working with a so-called line, which can be regarded as a known specific special case of the selection of clones.
  • sensitization The treatment of the cells with stimulation agents such as antigens or T-cell mitogens for the production of the selected clones used as the starting material is usually called sensitization here, while the further treatment of the selected cell clones with mitogenic stimulants is called stimulation here. It can in both cases, of course, treatment with the same stimulation agents under the same conditions, for example concanavalin A or phytohemagglutinin. Due to the different designation, only the stage according to the invention (stimulation) should be distinguished from the known preliminary stage (sensitization).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

1. Process for the preparation of type II interferon from T lymphocytes by sensitising and selecting clones from heterogeneous T lymphocyte populations, characterised in that these cell clones are subjected to mitogenic stimulation and, after further culturing of the stimulated clones, type II interferon is harvested in particular from the cell-free supernatants of the cultures.

Description

Erfindungsgegenstand ist ein Verfahren zur Herstellung von Typ II Interferon aus T-Lymphozyten durch Sensibilisierung und Selektionierung von Klonen aus heterogenen T-Lymphozyten-Populationen, dadurch gekennzeichnet, dass diese Zellklone einer mitogenen Stimulation unterworfen werden und nach Weiterzüchtung der stimulierten Klone Typ II Interferon, insbesondere aus den zellfreien Überständen der Kulturen, geerntet wird.The subject of the invention is a process for the production of type II interferon from T lymphocytes by sensitization and selection of clones from heterogeneous T lymphocyte populations, characterized in that these cell clones are subjected to mitogenic stimulation and after further growth of the stimulated clones type II interferon, in particular is harvested from the cell-free supernatants of the cultures.

Weiterer Gegenstand sind Typ II Interferon produzierende Zellklone zur Durchführung dieses Verfahrens die aus T-Lymphozyten durch Sensibilisierung und Selektionierung von Klonen aus heterogenen T-Lymphozyten-Populationen erhalten sind, dadurch gekennzeichnet, dass diese Zellklone danach einer mitogenen Stimulation unterworfen sind.Another subject are type II interferon-producing cell clones for performing this method, which are obtained from T-lymphocytes by sensitization and selection of clones from heterogeneous T-lymphocyte populations, characterized in that these cell clones are then subjected to mitogenic stimulation.

Interferon wird von körpereigenen Zellen produziert und hat in der letzten Zeit erhöhte Aufmerksamkeit gewonnen, da es die Neusynthese von Viren hemmt. Es wird auch diskutiert, ob Interferon nicht nur bei Virus- sondern auch bei Tumorerkrankungen eine therapeutische Wirkung erzielt, und es gibt Gründe, dies zu bejahen.Interferon is produced by the body's own cells and has recently received increased attention as it inhibits the resynthesis of viruses. It will also discusses whether interferon has a therapeutic effect not only in viral but also in tumor diseases, and there are reasons to say so.

Anhand biologischer und biochemischer Parameter unterscheidet man zwei Typen von Interferon, Typ I und Typ II. Typ I wird in Zellen unter anderem nach Virusinfektionen produziert, z.B. aus Leukozyten und Fibroblasten sowie Lymphoblasten (Kirchner et al., Pharmacie in unserer Zeit, 8, 113,1979). Es gibt Grund zur Annahme, dass TypII Interferon aus Immunozyten erhältlich ist (Kirchner et al., Immunobiology 156, 65, 1979 und Kirchner et al., Eur. J. Immunol. 10: 224, 1980).Based on biological and biochemical parameters, a distinction is made between two types of interferon, type I and type II. Type I is produced in cells after virus infections, e.g. from leukocytes and fibroblasts as well as lymphoblasts (Kirchner et al., Pharmacie in our time, 8, 113, 1979). There is reason to believe that type II interferon is obtainable from immunocytes (Kirchner et al., Immunobiology 156, 65, 1979 and Kirchner et al., Eur. J. Immunol. 10: 224, 1980).

In Tiermodellen gibt es Hinweise dafür, dass Typ II Interferon gegen Tumoren erheblich wirksamer ist als Typ I Interferon.In animal models, there is evidence that type II interferon is significantly more effective against tumors than type I interferon.

Obwohl die erste Arbeit über Interferon 1957 veröffentlicht wurde, ist die Herstellung immer noch ein Problem. Eine wesentliche Voraussetzung für den experimentellen und klinischen Einsatz von Interferon jedoch ist seine Reindarstellung basierend auf der Produktion in ausreichender Quantität. Dafür gibt es bisher für Typ II Interferon keine zufriedenstellende Möglichkeit.Although the first work on interferon was published in 1957, manufacturing is still a problem. An essential prerequisite for the experimental and clinical use of interferon, however, is its pure presentation based on the production in sufficient quantity. So far, there has been no satisfactory way of doing this for type II interferon.

Der Erfindungsgegenstand liefert nun ein Verfahren zur Herstellung von Typ II Interferon in grossem Masstab und gestattet die Erzielung von erhöhten Konzentrationen an Typ II Interferon in den bei der Herstellung anfallenden Lösungen.The subject of the invention now provides a process for the production of type II interferon on a large scale and permits the attainment of increased concentrations of type II interferon in the solutions obtained in the production.

Das nun gefundene.Verfahren und die stimulierten Klone können die Basis für eine industrielle Grossproduktion von Typ II Interferon darstellen.The process now found and the stimulated clones can form the basis for large-scale industrial production of type II interferon.

Wichtig bei diesem Verfahren ist, dass es sich im Gegensatz zu verschiedenen bei Typ I angewandten Verfahren bei den Interferon produzierenden Zellen um normale Zellen und nicht um Tumorzellen handelt, wobei die gestellte Aufgabe im Wesentlichen dadurch gelöst wird, dass Klone aus heterogenen Populationen selektioniert und die ausgewählten Klone mit mitogenen Stimulantien nachstimuliert werden.It is important in this method that, in contrast to various methods used in type I, the interferon-producing cells are normal cells and not tumor cells, the object being achieved essentially by selecting clones from heterogeneous populations and the selected clones can be stimulated with mitogenic stimulants.

Es ist überraschend, dass man hier einen Erfolg erzielt, da man annahm, dass diese Zellklone nicht mehr stimulierbar wären.It is surprising that success was achieved here, since it was assumed that these cell clones could no longer be stimulated.

Gemäss einer bevorzugten Ausführungsform werden aus den Einzelklonen homogene Massenkulturen gezüchtet und diese T-Zellklone einer mitogenen Stimulation unterworfen.According to a preferred embodiment, homogeneous mass cultures are grown from the individual clones and these T cell clones are subjected to mitogenic stimulation.

Gemäss weiteren bevorzugten Ausführungsformen erfolgt die mitogene Stimulation der T-Zellklone mit verschiedenen T-Zellmitogenen oder es werden verschiedene Konzentrationen des mitogenen Stimulationsmittels angewandt.According to further preferred embodiments, the mitogenic stimulation of the T cell clones is carried out with different T cell mitogens or different concentrations of the mitogenic stimulating agent are used.

Nach einer weiteren bevorzugten Ausführungsform werden die nach der Stimulation erhaltenen Zellklone separat weitergezüchtet.According to a further preferred embodiment, the cell clones obtained after the stimulation are cultivated separately.

Nach einer weiteren bevorzugten Ausführungsform erfolgt die Stimulation der Zellklone in serumfreiem Medium.According to a further preferred embodiment, the cell clones are stimulated in serum-free medium.

Es ist auch bevorzugt, das zur Interferon-Produktion benötigte genetische Material der selektionierten Klone in-an sich bekannter Weise in Bakterien oder Tumorzellen zur Gewinnung von Interferon zu benutzen.It is also preferred to use the genetic material of the selected clones required for interferon production in a manner known per se in bacteria or tumor cells to obtain interferon.

T-Lymphozyten (Thymus abh. Lymphozyten) aus lymphoiden Organen (Lymphknoten oder Milz) oder dem Blut werden in Gewebekultur sensibilisiert. Diese Sensibilisierung kann z.B. durch Antigene oder T-Zellmitogene, wie Concanavalin A oder Phytohämagglutinin erfolgen. Als günstig hat sich vor allem Concanavalin A in der Konzentration von 5 µg/ml bei einer Aussaat.von 5 x 106 Lymphknotenzellen/ml (4 ml Kulturen) erwiesen.T-lymphocytes (thymus-dependent lymphocytes) from lymphoid organs (lymph nodes or spleen) or the blood are sensitized in tissue culture. This sensitization can take place, for example, through antigens or T cell mitogens, such as concanavalin A or phytohemagglutinin. Concanavalin A in a concentration of 5 µg / ml has proven to be particularly favorable when seeding 5 x 10 6 lymph node cells / ml (4 ml cultures).

Die sensibilisierten T-Lymphozyten werden nach einer 2- bis 3-Tage dauernden Kultur gereinigt und mit Wachstumsfaktoren versetzt. Diese Wachstumsfaktoren werden aus dem Kulturüberstand von z.B. mit 5 µg Cöncanavalin A/ml sensibilisierten Rattenmilzlymphozyten (5.x 106 Zellen/ml, 50 ml Kulturen) gewonnen. Dieses Verfahren führt dazu, dass einige T-Lymphozyten als normale Zellen kontinuierlich zu wachsen beginnen. Das kontinuierliche Wachstum der T-Lymphozyten ist abhängig von der Präsenz und der Menge der Wachstumsfaktoren in der Kultur. Solche T-Lymphozytenzellinien werden dann in Mikrotiterplatten unter-Zugabe von bestrahlten Fütterzellen (z.B. Peritonealexudatzellen der Maus) kloniert. Aus den Einzelklonen werden homogene Massenkulturen als Nachkommen von Einzelzellen erhalten, die in grösseren Flaschen weiterkultiviert werden können und kontinuierlich wachsen. Diese Klone repräsentieren ein Spektrum von funktionell heterogenen normalen T-Lymphozyten in permanenter Gewebekultur.The sensitized T lymphocytes are cleaned after a 2 to 3 day culture and growth factors are added. These growth factors are obtained from the culture supernatant of rat splenic lymphocytes sensitized with 5 μg Cöncanavalin A / ml (5.x 10 6 cells / ml, 50 ml cultures). This procedure causes some T lymphocytes to start growing continuously as normal cells. The continuous growth of T-lymphocytes depends on the presence and the amount of growth factors in the culture. Such T-lymphocyte cell lines are then cloned in microtiter plates with the addition of irradiated feed cells (eg mouse peritoneal cells). From the individual cloning, homogeneous mass cultures are obtained as descendants of single cells that can be cultivated further in larger bottles and grow continuously. These clones represent a spectrum of functionally heterogeneous normal T lymphocytes in permanent tissue culture.

Bisher wurden solche T-Zellklone entweder funktionell inaktiv gefunden, oder sie behielten die primär induzierte spezifische Aktivität, die z.B. durch Antigenstimulation induziert worden war, bei. Es war nicht bekannt, dass die T-Zellklone durch weitere mitogene Stimulation zur Expression von funktionellen Aktivitäten gebracht werden konnten. Insbesondere sind Versuche negativ. verlaufen, die zum Ziel hatten, in diesen T-Zellklonen durch Inkubation mit Mitogenen ein verstärkt proliferatives Wachstum zu erzeugen. Deswegen ist die Idee, durch Mitogenstimulation der T-Zellklone Interferon Typ II zu produzieren, nicht naheliegend sondern eine wesentliche Neuerung.So far, such T cell clones have either been found functionally inactive or they have retained the primarily induced specific activity, e.g. had been induced by antigen stimulation. It was not known that the T cell clones could be brought to the expression of functional activities by further mitogenic stimulation. Experiments in particular are negative. that had the goal of generating increased proliferative growth in these T cell clones by incubation with mitogens. Therefore, the idea of producing interferon type II by mitogen stimulation of the T cell clones is not an obvious one, but a major innovation.

Es ist erfindungsgemäss gelungen, durch weitere Stimulation solcher Klone mit verschiedenen T-Zellmitogenen die herauszuselektionieren, die hohe Titer.von Typ II Interferon produzieren. Dabei ist einmal von Bedeutung, dass klonierte Zellen in der Lage sind, Interferon Typ II zu sezernieren, zum anderen übertrifft die Menge des sezernierten Typ II Interferons die Quantität, die aus einer normalen Lymphozytenpopulation bereitgestellt wird, um ein Vielfaches. Dieses neue Verfahren erlaubt unabhängig von Speziesschranken a) die Definition von Typ II Interferon produzierenden Zellen und b) die Anreicherung von grossen Mengen von Zellen, die Typ II Interferon produzieren als Basis für die biochemische Reindarstellung für einen weiteren experimentellen und therapeutischen Einsatz.According to the invention, further stimulation of such clones with various T cell mitogens has made it possible to select those which produce high titers of type II interferon. It is important that cloned cells are able to secrete type II interferon, and secondly that the amount of type II interferon secreted exceeds the quantity provided by a normal lymphocyte population many times over. This new procedure allows independent of species barriers a) the definition of type II interferon producing cells and b) the enrichment of large amounts of cells that produce type II interferon as the basis for the pure biochemical representation for further experimental and therapeutic use.

Dieses Verfahren gestattet es, hohe Mengen an Interferon vom Typ II herzustellen; zusätzlich kann die Konzentration an Typ II Interferon in den gewonnenen Lösungen auf mehr als das 10-fache gegenüber Massenkulturen von gemischten Zellen erhöht werden, was nicht nur für die gewonnene absolute Menge sondern auch eine Reindarstellung oder Gewinnung von konzentrierten Präparaten von Bedeutung ist.This process allows large amounts of type II interferon to be produced; in addition, the concentration of type II interferon in the solutions obtained can be increased more than 10 times compared to mass cultures of mixed cells, which is important not only for the absolute amount obtained, but also for the pure presentation or extraction of concentrated preparations.

Die folgenden Beispiele erläutern die Erfindung:The following examples illustrate the invention:

Beispiel 1:Example 1:

AKR/J Mäuse wurden, wie beschrieben, Krammer et al., J. Exp. Med. 151: 1166,1980, mit 2 x 10 µl 30% Trinitrochlorbenzol in Aceton auf der Abdominalhaut sensibilisiert. 5 Tage nach der Sensibilisierung wurden die Lymphozyten der drainierenden axillären und inguinalen Lymphknoten in Kultur gebracht (5 x 106 Zellen/ml; 4 ml Kulturen; Falconflaschen No. 3013, aufrecht) und für 4-bis 5 Tage in einem 95% Luft- 5% C02Gemisch inkubiert. Dabei kommt es ohne Antigenrestimulierung zu einer T-Zellproliferation, mit maximaler Aktivität am Tag 4 bis 5.AKR / J mice were, as described, Krammer et al., J. Exp. Med. 151: 1166, 1980, sensitized to the abdominal skin with 2 × 10 μl of 30% trinitrochlorobenzene in acetone. 5 days after sensitization, the lymphocytes of the draining axillary and inguinal lymph nodes were brought into culture (5 × 10 6 cells / ml; 4 ml cultures; Falcon bottles No. 3013, upright) and for 4 to 5 days in a 95% air 5% C0 2 mixture incubated. This leads to a T cell proliferation without antigen restimulation feration, with maximum activity on day 4 to 5.

Die überlebenden Zellen wurden dann wie beschrieben (P.H. Krammer, J. Exp. Med. 147: 25, 1978) durch eine Ficolldichtezentrifugation (Ficoll-Hypaque 1.077g/cm3) gereinigt und kloniert. Bei der Klonierung wurde im statischen Mittel wie beschrieben, (Eichmann et al., J. Exp. Med. 152: 477, 1980) eine Zelle/Loch von 96 Loch-Platten (Falcon No. 3040) auf einem Rasen von 5 x 104 Fütterzellen (Peritonealexudatzellen der AKR/J Maus) ausgesät. Die Klonierung erfolgte in Gegenwart von 10% T-Zellwachstumsfaktorhaltigem Vollmedium.The surviving cells were then purified and cloned as described (PH Krammer, J. Exp. Med. 147: 25, 1978) by Ficoll density centrifugation (Ficoll-Hypaque 1,077 g / cm 3 ). When cloning, the static mean as described (Eichmann et al., J. Exp. Med. 152: 477, 1980) was a cell / hole of 96-well plates (Falcon No. 3040) on a 5 × 10 lawn 4 feed cells (peritoneal extrudate cells from the AKR / J mouse) sown. The cloning was carried out in the presence of full medium containing 10% T cell growth factor.

Der Wachstumsfaktorhaltige überstand wurde gewonnen von Rattenmilzlymphozyten, stimuliert in Gewebekultur für 24 Stunden mit Concanavalin A (5 x 106/ml; 50 ml; 5 µg Con A/ml; Falconflaschen No. 3024 F, liegend) (Eichmann et al., J. Exp. Med. 152: 477, 1980). Die Zellen in den 96-Loch-Platten wurden 2 mal/Woche mit frischem Medium gefüttert. Nach ca. 3 Wochen zeigten sich makroskopisch sichtbare T-Zellklone, die in grossen Flaschen (Falcon No. 3024 F) in demselben Medium weitergezüchtet wurden.The growth factor-containing supernatant was obtained from rat spleen lymphocytes, stimulated in tissue culture for 24 hours with concanavalin A (5 × 10 6 / ml; 50 ml; 5 μg Con A / ml; Falcon bottles No. 3024 F, lying) (Eichmann et al., J Exp. Med. 152: 477, 1980). The cells in the 96-well plates were fed twice a week with fresh medium. After about 3 weeks, macroscopically visible T cell clones were found, which were further grown in large bottles (Falcon No. 3024 F) in the same medium.

Jeweils 6 x 105 Zellen in 0,2 ml Medium der einzelnen Klone werden dann nach Waschen in die Löcher einer 96-Loch-Platte gebracht und mit verschiedenen Konzentrationen von Concanavalin A stimuliert. 24 Stunden später werden die zellfreien Überstände dieser Kulturen geernetet und in üblicher Weise auf Interferonaktivität getestet (Maus L Zellen, Vesikular Stomatitis Virus, Test der Hemmung der Virusneubildung) Es ist so gelungen, Klone von T Lymphozyten zu finden, die bis zu 10 000 IE/ml von Interferon produzieren. Nach akzeptierten physiokochemischen Kriterien handelte es sich hierbei um Typ II Interferon.6 × 10 5 cells each in 0.2 ml medium of the individual clones are then put into the holes of a 96-well plate after washing and stimulated with different concentrations of Concanavalin A. 24 hours later, the cell-free supernatants from these cultures are harvested and tested for interferon activity in the usual way (Mouse L cells, vesicular stomatitis virus, test of inhibition of new virus formation). It was thus possible to find clones of T lymphocytes which produce up to 10,000 IU / ml of interferon. According to accepted physiocochemical criteria, this was type II interferon.

Nach dieser Arbeitsweise ist es möglich, aus diesen Klonen Typ II Interferon in grosser Menge zu erzeugen.According to this procedure, it is possible to produce type II interferon in large quantities from these clones.

Beispiel 2:Example 2:

Dieses Beispiel ist ein Anwendungsbeispiel beim Menschen.This example is an application example in humans.

Aus dem peripheren Blut wurden Lymphozyten durch eine Ficolldichtezentrifugation getrennt. Die somit gewonnenen Zellen wurden mit Phythämagglutinin (PHA, 1%) aktiviert, nach 2 Tagen Kultur über Ficoll gereinigt und dann kloniert, indem im statistischen Mittel 1 bis 50 Zellen/ Loch von 96-Loch-Platten (Falcon Nr. 3040) auf einem Rasen von 2 x 104 bestrahlten Fütterzellen (Mausperitonealexudatzellen) und menschlichen peripheren Blutzellen (3 x 104) ausgesät wurde. Die Klonierung erfolgte in Gegenwart von 20% T-Zellwachstumsfaktor und 1% PHA-haltigem Vollmedium. Der Wachstumfaktor-haltige Überstand wurde gewonnen von menschlichen Lymphozyten stimuliert in Gewebekultur für 24 Stunden mit Phytohemagglutinin (5 x 106/ml; 50 ml; 1% PHA/ml; Falconflaschen Nr. 3024F, liegend). Die Zellen in den 96-Loch-Platten wurden 1 mal/Woche mit frischem Medium und Fütterzellen gefüttert. Nach ca. 3 Wochen zeigten sich makroskopisch sichtbare T-Zellklone.Lymphocytes were separated from peripheral blood by Ficoll density centrifugation. The cells thus obtained were activated with phythaemagglutinin (PHA, 1%), purified after 2 days of culture via Ficoll and then cloned by means of a statistical average of 1 to 50 cells / hole from 96-hole plates (Falcon No. 3040) on a Lawn from 2 x 10 4 irradiated feed cells (mouse peritoneal cells) and human peripheral blood cells (3 x 10 4 ) was sown. The cloning was carried out in the presence of 20% T cell growth factor and 1% full medium containing PHA. The growth factor-containing supernatant was obtained from human lymphocytes stimulated in tissue culture for 24 hours with phytohemagglutinin (5 x 10 6 / ml; 50 ml; 1% PHA / ml; Falcon bottles No. 3024F, lying). The cells in the 96-well plates were washed once a week with fresh medium and fed feeding cells. After about 3 weeks, macroscopically visible T cell clones were shown.

Zellen der einzelnen Klone in 0,2 ml wachstumsfaktorfreiem Medium wurden dann mit 20 µg/ml PHA stimuliert, z.B. gemäss Beispiel 1. Dieser Schritt kann, und zwar auch bei Anwendung auf menschliche Zellen, auch in serumfreiem Medium, z.B. RPMI 1640 durchgeführt werden. 24 Stunden später wurden'die zellfreien überstände dieser Kulturen geerntet und in üblicher Weise auf Interferonaktivität getestet. Die Kulturen mit hohem Interferontiter wurden dann in Wachstumsfaktor- und PHA-haltigem Vollmedium unter Anwesenheit von bestrahlten menschlichen Fütterzellen zu Makrokulturen hochgezüchtet.Cells of the individual clones in 0.2 ml growth factor-free medium were then stimulated with 20 µg / ml PHA, e.g. according to example 1. This step can, even when applied to human cells, also in serum-free medium, e.g. RPMI 1640 can be performed. 24 hours later, the cell-free supernatants from these cultures were harvested and tested for interferon activity in the usual way. The cultures with high interferon titer were then grown to macro cultures in growth medium and PHA-containing complete medium in the presence of irradiated human feed cells.

Es ergaben sich Interferontiter.von mehreren hundert IE/ml.Interferon titers of several hundred IU / ml were found.

Die grosse Menge an Typ II Interferon produzierenden Zellen gestattet es aber auch,.die Interferon liefernden Gene bzw. das zur Interferonproduktion benutzte genetische Material der selektionierten Klone in an sich bekannter Weise in Bakterien oder Tumorzellen (in Gewebekultur) zur Gewinnung von Interferon zu benutzen, was ein zusätzliches Verfahren zur grosstechnischen Produktion von Typ II Interferon ermöglicht. Nachdem die Isolierung von Genen und der Einbau in Bakterien bzw. Tumorzellen bzw. die Verwendung von genetischem Material zur entsprechenden Veränderung von Bakterien oder Zellen als solche bekannt sind (erstere Arbeitsweise ist schon für Typ I Interferon erfolgreich versucht worden) bedarf diese spezielle Ausführungsform keinerlei weiteren Erläuterung.However, the large amount of type II interferon-producing cells also makes it possible to use the interferon-producing genes or the genetic material of the selected clones used for interferon production in a manner known per se in bacteria or tumor cells (in tissue culture) for obtaining interferon, which enables an additional process for the large-scale production of type II interferon. After the isolation of genes and the incorporation into bacteria or tumor cells or the use of genetic material for the corresponding modification of bacteria or cells are known as such (the former way of working is already this type of embodiment requires no further explanation.

Bei der Nachstimulierung hat sich beim Arbeiten in serumfreiem Medium, bei menschlichen Zellen, eine Bevorzugung für Phytohemagglutinin (PHA) ergeben, jedoch ist z.B. auch Concanavalin A brauchbar.During post-stimulation, preference has been given to phytohemagglutinin (PHA) when working in serum-free medium, in human cells, but e.g. Concanavalin A can also be used.

Als Konzentrationen bei der Stimulation der Klone hat sich wie bei der bekannten Sensibilisierung (= Stimulation der T-Lymphozyten) zur Herstellung von Klonen eine Menge von 5 bis 30 µg/ml, insbesondere 15 bis 25, vorzugsweise 10 bis 20 µg/ml als zweckmässig erwiesen.As in the known sensitization (= stimulation of the T-lymphocytes) for the production of clones, an amount of 5 to 30 μg / ml, in particular 15 to 25, preferably 10 to 20 μg / ml, has been found to be expedient as concentrations in the stimulation of the clones proven.

Der Begriff Klone ist selbstverständlich in breitem Umfang zu verstehen und umfasst auch das Arbeiten mit einer sogenannten Linie, die ja als bekannter spezifischer Sonderfall der Selektionierung von Klonen betrachtet werden kann.The term clones is, of course, to be understood in a broad sense and also includes working with a so-called line, which can be regarded as a known specific special case of the selection of clones.

Zur Terminologie, die hier benutzt wird, sei folgendes ausgeführt:Regarding the terminology used here, the following should be stated:

Die Behandlung der Zellen mit Stimulationsmitteln, wie Antigenen oder T-Zellmitogenen zur Herstellung der als Ausgangsmaterial verwendeten selektionierten Klone wird hier meist Sensibilisierung genannt, während die weitere Behandlung der selektionierten Zellklone mit mitogenen Stimulantien hier Stimulation genannt wird. Es kann sich natürlich in beiden Fällen um die Behandlung mit den gleichen Stimulationsmitteln unter den gleichen Bedingungen handeln, z.B. Concanavalin A oder Phytohemagglutinin. Es soll durch die unterschiedliche Bezeichnung nur die Stufe gemäss der Erfindung (Stimulation) von der bekannten Vorstufe (Sensibilisierung) unterschieden werden.The treatment of the cells with stimulation agents such as antigens or T-cell mitogens for the production of the selected clones used as the starting material is usually called sensitization here, while the further treatment of the selected cell clones with mitogenic stimulants is called stimulation here. It can in both cases, of course, treatment with the same stimulation agents under the same conditions, for example concanavalin A or phytohemagglutinin. Due to the different designation, only the stage according to the invention (stimulation) should be distinguished from the known preliminary stage (sensitization).

Claims (8)

1. Verfahren zur Herstellung von Typ II.Interferon aus T-Lymphozyten durch Sensibilisierung und Selektionierung von Klonen aus heterogenen T-Lymphozyten-Populationen, dadurch gekennzeichnet, dass diese Zellklone einer mitogenen Stimulation unterworfen werden und nach Weiterzüchtung der stimulierten Klone Typ II Interferon, insbesondere aus den zellfreien überständen der Kulturen, geerntet wird.1. A process for the production of type II. Interferon from T lymphocytes by sensitization and selection of clones from heterogeneous T lymphocyte populations, characterized in that these cell clones are subjected to mitogenic stimulation and after further growth of the stimulated clones type II interferon, in particular harvested from the cell-free supernatants of the cultures. 2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass aus den Einzelklonen homogene Masserikulturen gezüchtet .und diese T-Zellklone einer mitogenen Stimulation unterworfen werden.2. The method according to claim 1, characterized in that homogeneous mass cultures are grown from the individual clones. And these T cell clones are subjected to mitogenic stimulation. 3. Verfahren nach einem der Ansprüche 1 oder 2, dadurch gekennzeichnet, dass die mitogene Stimulation der T-Zellklone mit verschiedenen T-Zellmitogenen erfolgt.3. The method according to any one of claims 1 or 2, characterized in that the mitogenic stimulation of the T cell clones is carried out with different T cell mitogens. 4. Verfahren nach Anspruch 1 bis 3, dadurch gekennzeichnet, dass verschiedene Konzentrationen des mitogenen Stimulationsmittels angewandt werden.4. The method according to claim 1 to 3, characterized in that different concentrations of the mitogenic stimulation agent are used. 5. Verfahren nach Anspruch 3 oder 4, dadurch gekennzeichnet, dass die nach der Stimulation erhaltenen Zellklone separat weitergezüchtet werden.5. The method according to claim 3 or 4, characterized in that the cell clones obtained after the stimulation are grown separately. 6. Verfahren nach einem oder mehreren der vorhergehenden Ansprüche 1 bis 5, dadurch gekennzeichnet, dass die Stimulation in serumfreiem Medium durchgeführt wird, z.B. RPMI Medium.6. The method according to one or more of the preceding claims 1 to 5, characterized in that the stimulation is carried out in serum-free medium, e.g. RPMI medium. 7. Verfahren nach einem oder mehreren der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass das zur Interferon-Produktion benötigte genetische Material der selektionierten Klone in an sich bekannter Weise in Bakterien oder Tumorzellen zur Gewinnung von Interferon benutzt wird.7. The method according to one or more of the preceding claims, characterized in that the genetic material of the selected clones required for interferon production is used in a manner known per se in bacteria or tumor cells for the production of interferon. 8. Typ II Interferon produzierende Zellklone zur Durchführung des Verfahrens nach Anspruch 1 bis 7, die aus T-Lymphozyten durch Sensibilisierung und Selektionierung von Klonen aus heterogenen T-Lymphozyten Populationen erhalten sind, dadurch gekennzeichnet, dass diese Zellklone danach einer mitogenen Stimulation unterworfen sind.8. Type II interferon-producing cell clones for performing the method according to claim 1 to 7, which are obtained from T-lymphocytes by sensitization and selection of clones from heterogeneous T-lymphocyte populations, characterized in that these cell clones are then subjected to mitogenic stimulation.
EP81103918A 1980-05-22 1981-05-21 Process for the production of interferon ii and stimulated clones for carrying out the process Expired EP0041189B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AT81103918T ATE3877T1 (en) 1980-05-22 1981-05-21 METHOD FOR PRODUCTION OF INTERFERON II AND STIMULATED CLONES FOR CARRYING OUT THE METHOD.

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE3019621 1980-05-22
DE3019621A DE3019621C2 (en) 1980-05-22 1980-05-22 Process for the production of interferon II.
DE19813104900 DE3104900A1 (en) 1981-02-11 1981-02-11 Process for the preparation of interferon II and stabilised clones for carrying out the process
DE3104900 1981-02-11

Publications (2)

Publication Number Publication Date
EP0041189A1 true EP0041189A1 (en) 1981-12-09
EP0041189B1 EP0041189B1 (en) 1983-06-22

Family

ID=25785623

Family Applications (1)

Application Number Title Priority Date Filing Date
EP81103918A Expired EP0041189B1 (en) 1980-05-22 1981-05-21 Process for the production of interferon ii and stimulated clones for carrying out the process

Country Status (6)

Country Link
EP (1) EP0041189B1 (en)
AU (1) AU7086781A (en)
DE (1) DE3160481D1 (en)
DK (1) DK220781A (en)
GR (1) GR74132B (en)
IL (1) IL62903A0 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992018538A1 (en) * 1991-04-11 1992-10-29 Tomas Olsson A lymphocyte stimulating factor originating from the haemoflagellate trypanosoma
US5891439A (en) * 1996-07-26 1999-04-06 Sbl Vaccin Ab, Stockholm, Se Lymphocyte stimulating factor
US7588755B1 (en) 1980-04-03 2009-09-15 Biogen Idec Ma Inc. DNA sequences, recombinant DNA molecules and processes for producing human fibroblast interferon-like polypeptides
US8591956B2 (en) 2007-11-28 2013-11-26 Irx Therapeutics, Inc. Method of increasing immunological effect
US8784796B2 (en) 2000-10-27 2014-07-22 Irx Therapeutics, Inc. Vaccine immunotherapy for treating hepatocellular cancer in immune suppressed patients
US9333238B2 (en) 2009-12-08 2016-05-10 Irx Therapeutics, Inc. Method of immunotherapy for treament of human papillomavirus infection
US9492517B2 (en) 2000-10-27 2016-11-15 Irx Therapeutics, Inc. Vaccine immunotherapy
US9539320B2 (en) 2009-05-15 2017-01-10 Irx Therapeutics, Inc. Vaccine immunotherapy

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2352662A1 (en) * 1972-10-20 1974-05-02 Piktor Ltd METHOD FOR PRODUCING A TRANSFER FACTOR
DE2461379A1 (en) * 1974-01-11 1975-07-17 Anvar PROCESS FOR MANUFACTURING INTERFERON IN LARGE QUANTITIES AND ON AN INDUSTRIAL SCALE
FR2257306A1 (en) * 1974-01-11 1975-08-08 Anvar Interferon large-scale production - involving simulation with interferon and induction by incubating with virus

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2352662A1 (en) * 1972-10-20 1974-05-02 Piktor Ltd METHOD FOR PRODUCING A TRANSFER FACTOR
FR2203622A1 (en) * 1972-10-20 1974-05-17 Piktor Ltd
DE2461379A1 (en) * 1974-01-11 1975-07-17 Anvar PROCESS FOR MANUFACTURING INTERFERON IN LARGE QUANTITIES AND ON AN INDUSTRIAL SCALE
FR2257306A1 (en) * 1974-01-11 1975-08-08 Anvar Interferon large-scale production - involving simulation with interferon and induction by incubating with virus

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Arch. Intern Med., Band 126, Juli 1970, Chigago (US) E. DE CLERCO et al.: "Induction of Interferon by Nonviral Agents, seiten 94-108 * seite 95, linke spalte, zeile 50 bis Mittele spalte, zeile 9; seite 97, rechte spalte, zeilen 38-48; seite 101: "Multiple Effects of Interferon...", bis seite 102, linke spalte, zeile 25 * *
CHEMICAL ABSTRACTS, Band 67, Heft 15, 9. Oktober 1967, seite 6764, Zusammenfassung 71985u, Columbus, Ohio (US) R.M. FRIEDMAN et al.: "Stimulation of Interferon Production in Human Lymphocytes by Mitogens" & Proc. Soc. Exp. Biol. Med. 125 (3), 901-5 (1967) * ingesamt * *
Nature, Band 280, Heft 5724, August 1979, Basingstoke (US) J.M. ZARLING et al.: "Continuous Culture of T Cells Cytotoxix for Autologous Human Leukaemia Cells" seiten 685-688 * seiten 685, zeile 18 bis seite 686, linke spalte, zeile 3 * *
Nature, Band 283, Heft 5745, Januar 1980, Basingstoke (US) P. Newmark: "Engineering E. Coli Produce Interferon", seite 323 * ingesamt * *
The Journal of Immunology, Band 112 Heft 2, Februar 1974, Baltimore (US) L.B. EPSTEIN et al.: "Evaluation of T Lymphocyte Effector Function in Immunodeficiency Diseases: Abnormality in Mitogen-Stimulated Interferon in Patients with Selective IgA Deficiency" * seite 623: "Discussion" bis Ende der seite * *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7588755B1 (en) 1980-04-03 2009-09-15 Biogen Idec Ma Inc. DNA sequences, recombinant DNA molecules and processes for producing human fibroblast interferon-like polypeptides
US7635466B1 (en) 1980-04-03 2009-12-22 Biogen Idec Ma Inc. DNA sequences, recombinant DNA molecules and processes for producing human fibroblast interferon-like polypeptides
WO1992018538A1 (en) * 1991-04-11 1992-10-29 Tomas Olsson A lymphocyte stimulating factor originating from the haemoflagellate trypanosoma
US5891439A (en) * 1996-07-26 1999-04-06 Sbl Vaccin Ab, Stockholm, Se Lymphocyte stimulating factor
US9789173B2 (en) 2000-10-27 2017-10-17 Irx Therapeutics, Inc. Vaccine immunotherapy for treating cervical cancer in immune suppressed patients
US8784796B2 (en) 2000-10-27 2014-07-22 Irx Therapeutics, Inc. Vaccine immunotherapy for treating hepatocellular cancer in immune suppressed patients
US9789172B2 (en) 2000-10-27 2017-10-17 Irx Therapeutics, Inc. Vaccine immunotherapy for treating lymphoma in immune suppressed patients
US9492517B2 (en) 2000-10-27 2016-11-15 Irx Therapeutics, Inc. Vaccine immunotherapy
US9492519B2 (en) 2000-10-27 2016-11-15 Irx Therapeutics, Inc. Vaccine immunotherapy
US8591956B2 (en) 2007-11-28 2013-11-26 Irx Therapeutics, Inc. Method of increasing immunological effect
US9539320B2 (en) 2009-05-15 2017-01-10 Irx Therapeutics, Inc. Vaccine immunotherapy
US9566331B2 (en) 2009-05-15 2017-02-14 Irx Therapeutics, Inc. Vaccine immunotherapy
US9333238B2 (en) 2009-12-08 2016-05-10 Irx Therapeutics, Inc. Method of immunotherapy for treament of human papillomavirus infection
US9931378B2 (en) 2009-12-08 2018-04-03 Irx Therapeutics, Inc. Method of immunotherapy for treatment of human papillomavirus infection

Also Published As

Publication number Publication date
DK220781A (en) 1981-11-23
EP0041189B1 (en) 1983-06-22
DE3160481D1 (en) 1983-07-28
GR74132B (en) 1984-06-06
IL62903A0 (en) 1981-07-31
AU7086781A (en) 1981-11-26

Similar Documents

Publication Publication Date Title
DE69833649T2 (en) EF-1-ALPHA TRANSCRIPTIONAL REGULATORY DNA FROM HAMSTER
DE3043981C2 (en)
DE68928914T2 (en) (IN VITRO) PROPAGATION OF EMBRYONIC STEM CELLS USING LEUKEMIA INHIBITION FACTOR (LIF)
DE2651685C2 (en) Cultivation of keratinocytes
DE2902136A1 (en) INTERFERON AND PREPARATIONS CONTAINING IT
EP0372502A2 (en) Use of a bacterolytic enzyme and protease as an additive for feed conversion in animal rearing
EP0041189B1 (en) Process for the production of interferon ii and stimulated clones for carrying out the process
DE3249567C2 (en) Hybrid cell line, process for its production and its use
EP0005476A2 (en) Process for preparing human interferon
DE2757169A1 (en) METHOD OF OBTAINING INSULIN-PRODUCING ANIMAL CELLS
DE69832662T2 (en) Process for the culture of hepatocytes
DE3019621C2 (en) Process for the production of interferon II.
DE3325131C2 (en)
DE3048289A1 (en) "METHOD FOR BREEDING INSECT CELLS"
DE4244437A1 (en) Process for obtaining the body's own cytokines
EP0075904B1 (en) Process for culturing human fibroblasts and fibroblast-like cells
Smith Extreme chromosomal polymorphism in a coccinellid beetle
EP1034271A2 (en) Transgenic non-human mammals with an oncogenic mutant of raf gene 1
DE2821466A1 (en) Interferon prodn. - using stimulators to increase yields
DE2946275C2 (en)
DE3907679A1 (en) METHOD FOR TARGETED CHANGING A GENE
DE3104900A1 (en) Process for the preparation of interferon II and stabilised clones for carrying out the process
DE69022121T2 (en) Process for the preparation of a metastasis inhibitor factor.
DE3331860A1 (en) Process for the preparation of tendamistat
EP0075886A2 (en) Process for obtaining macrophage-activating factor

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Designated state(s): AT BE CH DE FR GB IT LU NL SE

17P Request for examination filed

Effective date: 19820309

ITF It: translation for a ep patent filed
GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Designated state(s): AT BE CH DE FR GB IT LI LU NL SE

REF Corresponds to:

Ref document number: 3877

Country of ref document: AT

Date of ref document: 19830715

Kind code of ref document: T

REF Corresponds to:

Ref document number: 3160481

Country of ref document: DE

Date of ref document: 19830728

ET Fr: translation filed
PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Effective date: 19840521

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Effective date: 19840522

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 19840531

Ref country code: LI

Effective date: 19840531

Ref country code: CH

Effective date: 19840531

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: BE

Payment date: 19840630

Year of fee payment: 4

26N No opposition filed
PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Effective date: 19841201

NLV4 Nl: lapsed or anulled due to non-payment of the annual fee
PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 19850131

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Effective date: 19850201

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Effective date: 19850531

BERE Be: lapsed

Owner name: STIFTUNG DEUTSCHES KREBSFORSCHUNGSZENTRUM

Effective date: 19850521

GBPC Gb: european patent ceased through non-payment of renewal fee
PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Effective date: 19881118

EUG Se: european patent has lapsed

Ref document number: 81103918.9

Effective date: 19850417